Composition, kit, and method for diagnosing and treating ovarian cancer

ABSTRACT

Provided are a composition, a kit, and a method of predicting prognosis of ovarian cancer or a risk of recurrence of ovarian cancer. Provided are a composition for treating ovarian cancer or preventing recurrence of ovarian cancer and a method of screening a material for treating ovarian cancer or preventing recurrence of ovarian cancer. According to the present disclosure, prognosis or recurrence of ovarian cancer can be efficiently diagnosed, and a candidate material that can treat ovarian cancer or prevent recurrence of ovarian cancer can be efficiently screened.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of Korean Patent Application No.10-2015-0169286, filed on Nov. 30, 2015, in the Korean IntellectualProperty Office, the disclosure of which is incorporated herein in itsentirety by reference.

BACKGROUND

1. Field

One or more embodiments relate to compositions, kits, and methods forpredicting prognosis of ovarian cancer or a risk of recurrence ofovarian cancer, and more particularly, to compositions for treatingovarian cancer or preventing recurrence of ovarian cancer and methods ofscreening a material for treating ovarian cancer or preventingrecurrence of ovarian cancer.

2. Description of the Related Art

Ovarian carcinoma is a cancer that has the highest lethality amongfemale malignancies, and this high lethality results fromchemoresistance and frequent recurrence of ovarian carcinoma. Severalagents have been developed to prevent or treat ovarian carcinoma, butthe mortality and rate of recurrence of ovarian carcinoma are stillhigh.

Recently, interest in cancer stem cells (CSCs), which exist in smallnumbers within tumors and have high tumorigenic capacity, has beenincreasing. CSCs were initially isolated from ovarian serous carcinomas(OSCs) by a spheroid formation assay, and are presumed to be one of themajor causes of recurrence and chemoresistance of cancers. However, noeffective strategies for reducing CSCs and lowering the high recurrenceand mortality rates of ovarian cancer have been developed yet.

SUMMARY

One or more embodiments include a composition for diagnosing prognosisof ovarian cancer or a risk of recurrence of ovarian cancer of anindividual.

One or more embodiments include a kit for diagnosing prognosis ofovarian cancer or a risk of recurrence of ovarian cancer of anindividual.

One or more embodiments include a method of diagnosing prognosis ofovarian cancer or a risk of recurrence of ovarian cancer of anindividual.

One or more embodiments include a composition for treating ovariancancer or preventing recurrence of ovarian cancer.

One or more embodiments include a method of screening a material fortreating ovarian cancer or preventing recurrence of ovarian cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

These and/or other aspects will become apparent and more readilyappreciated from the following description of the embodiments, taken inconjunction with the accompanying drawings in which:

FIGS. 1A to 1C relate to cancer stem cells (CSCs) isolated from culturedprimary cells from 17 ovarian serous carcinomas (OSCs) by a spheroidformation assay; and FIG. 1A shows (a) low view, (b) high view, and (c)power view phase-contrast images of primary cultures of OSC cells withspheroid morphology; FIG. 1B is a graph showing consistentspheroid-forming capacity of the primary cancer cells between the firstand second generations (2.17%-2.37%), indicating that thespheroid-forming cells (SFCs) exhibit self-renewing characteristics; andFIG. 1C is a graph showing significant expression of stem cell markersdetected in SFCs (*P<0.05) (known markers of stem cells are used, andall markers, except CD117, are significantly overexpressed in SFCs,indicating that the SFCs are enriched in CSCs as compared with thecultured primary cells);

FIGS. 2A to 2C shows results of cDNA microarray analysis of OSC SFCs andthe corresponding primary cancer cells; and FIG. 2A shows resultsrepresenting hierarchical clustering of significantly altered genes withat least 15-fold differences between SFCs and primary cancer cells,wherein the clustered expression data are displayed on a heat map withindividual genes listed on the X-axis and Y-axis, the gray-scale depictsrelative levels of gene expression in SFC samples compared with theircorresponding primary cancer cells from light (for downregulation) todark (for upregulation), and 28 genes were highly expressed in all fourSFC samples as compared with control cells while 34 genes weredownregulated in all four SFC samples as compared with control cells;FIG. 2B is a pie chart showing ontology of genes upregulated more than2-fold in SFCs (P<0.05), wherein genes associated with apoptosis andproliferation account for 13%, genes associated with cell cycle accountfor 10%, genes associated with signal transduction and CSCs account for8%, and genes associated with drug response account for 5%; and FIG. 2Cis a graph showing validation by quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR) of genes that are significantlyupregulated by cDNA microarray, wherein the qRT-PCR is performed on 17sets of SFCs and parental cancer cells, and GSC (4.26-fold), VAV3(7.05-fold), FOXA2 (12.06-fold), LEF1 (17.26-fold), COMP (21.33-fold),GRIN2A (9.36-fold), CD86 (23.14-fold), PYY (4.18-fold), NKX3-2(10.35-fold), and PDK4 (74.26-fold) are significantly upregulated inSFCs as compared with parental cancer cells thereof (*P<0.05);

FIG. 3A is a graph showing mRNA expression levels of genes selected byqRT-PCR, wherein the mRNA expression levels of 13 genes aresignificantly increased in SFCs as compared with those of primary cancercells, and 11 genes are significantly overexpressed in OSCs as comparedwith control fallopian tubes (*P<0.05); FIG. 3B is a graph indicatingthat the relative mRNA levels of LEF1, PYY, NKX3-2, and WNT3A genes aresignificantly increased in chemoresistant cancer cells as compared withthose in chemosensitive cancer cells; and FIG. 3C is a graph showingassociations between clinicopathologic parameters and the mRNAexpression levels identified by qRT-PCR, wherein (a) shows significantassociation between the expression level of PYY (≧2-fold increase) andlymph node metastasis (*P<0.05), (b) shows significant associationbetween the expression level of VAV3 (≧2-fold increase) and distantmetastasis (*P<0.05), and (c) shows association between the expressionlevels of PYY (2-fold increase), FOXA2 (≧6-fold increase), and NKX3-2(10-fold increase) and increased chemoresistance in OSCs (*P<0.05);

FIG. 4A are images showing immunohistochemical staining of OSCs andcontrol tissues, wherein (a,b) indicate positive nuclear staining forFOXA2 in OCSs but negative staining in fallopian tubes, (c,d) indicatepositive nuclear staining for LEF1 in OSCs, but negative staining infallopian tubes, (e,f) indicate positive nuclear and cytoplasmicreactivity for VAV3 in OCSs, but negative staining in fallopian tubes,(g,h) indicate NKX3-2 staining in the nuclei of OSCs, but no staining infallopian tubes, (i,j) indicate membranous staining for Wht3A in OSCs,but only luminal staining in fallopian tubes, and (k,l) indicatePYY-positive staining in OSCs, but negative staining in fallopian tubes;and FIG. 4B shows survival curves according to VAV3 and FOXA2immunohistochemical reactivity, indicating that patients with highlevels of VAV3 (a) or FOXA2 (b) expression show poorer survival thanpatients with low VAV3 or FOXA2 expression (P<0.05); and

FIGS. 5A to 5C show effects of VAV3 knockdown on paclitaxel(PTX)-resistant ovarian cancer cells; and FIG. 5A shows results of aspheroid formation assay, wherein the number of spheroid formationsdecreases by 30% after treatment of VAV3 siRNA as compared with that ofspheroid formations in a negative siRNA control, the size of spheroidsis significantly decreased in VAV3 siRNA-treated SKpac cells as comparedwith that of spheroids in the negative siRNA control, and the graphrepresents the mean±standard error (*P<0.001); FIG. 5B shows results ofa colony formation assay, wherein VAV3 knockdown cells are seeded at 300cells per well and cultured for 14 days, and consequently, the number ofcolonies of VAV3 siRNA-treated SKpac cells decreases by 41% as comparedwith control cells treated with negative siRNA, and the graph representsthe mean±standard error of triplicate experiments (*P<0.001); and FIG.5C shows results of an MTT assay, wherein cell viability is assessedafter VAV3 siRNA treatment, and consequently, cell viability decreasesby 25% and 18% (*P<0.05) at 24 hours and 48 hours, respectively, in VAV3siRNA-treated SKpac cells as compared with control cells treated withnegative siRNA, and upon treatment with VAV3 siRNA and 40 nM PTX, thenumber of SKpac cells decreases by 27% and 16% (*P<0.05) at 24 hours and48 hours, respectively, compared with that of control cells treated withPTX+negative siRNA, and the graph represents the mean±standard error oftriplicate experiments.

DETAILED DESCRIPTION

Reference will now be made in detail to embodiments, examples of whichare illustrated in the accompanying drawings, wherein like referencenumerals refer to like elements throughout. In this regard, the presentembodiments may have different forms and should not be construed asbeing limited to the descriptions set forth herein. Accordingly, theembodiments are merely described below, by referring to the figures, toexplain aspects of the present description.

According to one or more embodiments, there is provided a compositionfor predicting prognosis of ovarian cancer or a risk of recurrence ofovarian cancer of an individual, the composition including an agent formeasuring an expression level of one or more genes selected from thegroup consisting of GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F,CD86, PYY, FIGF, NKX3-2, WNT3A, and PDK4.

Goosecoid homeobox gene (GSC) may be a gene having an mRNA sequence ofNCBI Accession No: NM_173849.2. Vav 3 guanine nucleotide exchange factorgene (VAV3) may be a gene having an mRNA sequence of NCBI Accession No:NM_006113.4 or NM_001079874.1. Forkhead box A2 gene (FOXA2) may be agene having an mRNA sequence of NCBI Accession No: NM_021784.4 orNM_153675.2. Lymphoid enhancer-binding factor 1 gene (LEF1) may be agene having an mRNA sequence of NCBI Accession No: NM_016269.4,NM_001166119.1, NM_001130714.2, or NM_001130713.2. Cartilage oligomericmatrix protein gene (COMP) may be a gene having an mRNA sequence of NCBIAccession No: NM_000095.2. Azurocidin 1 gene (AZU1) may be a gene havingan mRNA sequence of NCBI Accession No: NM_001700.4. Glutamate receptor,ionotropic, N-methyl D-aspartate 2A gene (GRIN2A) may be a gene havingan mRNA sequence of NCBI Accession No: NM_001134408.2, NM_000833.4, orNM_001134407.2. Interleukin 17F gene (IL17F) may be a gene having anmRNA sequence of NCBI Accession No: NM_052872.3. CD86 molecule gene(CD86) may be a gene having an mRNA sequence of NCBI Accession No:NM_001206925.1, NM_001206924.1, NM_176892.1, NM_175862.4, orNM_006889.4. Peptide YY gene (PYY) may be a gene having an mRNA sequenceof NCBI Accession No: NM_004160.4. C-fos induced growth factor (vascularendothelial growth factor D) gene (FIGF) may be a gene having an mRNAsequence of NCBI Accession No: NM_004469.4. NK3 homeobox 2 gene (NKX3-2)may be a gene having an mRNA sequence of NCBI Accession No: NM_001189.3.Wingless-type MMTV integration site family member 3A gene (WNT3A) may bea gene having an mRNA sequence of NCBI Accession No: NM_033131.3.Pyruvate dehydrogenase kinase, isozyme 4 gene (PDK4) may be a genehaving an mRNA sequence of NCBI Accession No: NM 002612.3.

The inventors of the present inventive concept have found that increasedor decreased expression of GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A,IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A, or PDK4 is associated withprognosis of ovarian cancer or recurrence of ovarian cancer. In thisregard, an expression level of GSC, VAV3, FOXA2, LEF1, COMP, AZU1,GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A, or PDK4 may be consideredwhen diagnosing prognosis of ovarian cancer or a risk of recurrence ofovarian cancer.

GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF,NKX3-2, WNT3A, and PDK4, which are proteins used in the presentspecification, are construed as including respectively naturallyoccurring wild-type GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F,CD86, PYY, FIGF, NKX3-2, WNT3A, and PDK4, or functional variantsthereof. In addition, GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F,CD86, PYY, FIGF, NKX3-2, WNT3A, and PDK4, which are genes used in thepresent specification, are construed as respectively including naturallyoccurring wild-type genes of GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A,IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A, and PDK4, or functional variantsthereof.

The expression level may include an expression level of any gene capableof encoding GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86,PYY, FIGF, NKX3-2, WNT3A, or PDK4. The expression level may also referto an expression level of a material at an mRNA or protein stage.Therefore, the composition may include an agent in terms of measuringamounts of mRNA, protein, or a combination thereof, of GSC, VAV3, FOXA2,LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A, orPDK4. In an example embodiment, the agent may specifically bind to atranscriptome of GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F,CD86, PYY, FIGF, NKX3-2, WNT3A, or PDK4. In various example embodiments,the agent may include a primer, a probe, or an antisense sequence, orcombinations of thereof, which specifically binds to mRNA of GSC, VAV3,FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A,or PDK4, or to a complementary sequence of the mRNA. In various exampleembodiments, the agent may include a primer, a probe, or an antisensesequence, or a combination thereof, which specifically binds to a geneencoding GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY,FIGF, NKX3-2, WNT3A, or PDK4. In various example embodiments, the agentmay be screened according to a method known in the art, such as a methodof high throughput screening (HTS), and in addition, may be commerciallyavailable.

The primer may serve as an initiation point for polymerization using apolymerase. The primer used herein may also be used in nucleic acidamplification. The term “amplification” as used herein may refer to anincrease of copy number of a target sequence or a complementary sequenceof the target sequence. The nucleic acid amplification may be performedusing any method known in the art. For example, the nucleic acidamplification may be performed using a method employing multiple cyclesduring the amplification or a method performed at a constanttemperature. For example, cycling techniques for the amplification mayrequire a heat cycle. An example of a method requiring a heat cycleincludes polymerase chain reaction (PCR), which is known in the art. PCRgenerally includes: thermally denaturing double-stranded DNA to producesingle-stranded DNA; annealing a primer to the single-stranded DNA; andsynthesizing a complementary strand from the primer. Isothermalamplification may be performed at a constant temperature or a majoraspect of the amplification process occurs at a constant temperature. Incontrast to PCR, which allows reaction products to be heated to bind toadditional primers to separate double strands, an isothermal method usesa strand displacing polymerase in a dependent manner to separate doublestrands and re-copy a template. Such an isothermal method is differentfrom a method that relies on primer substitution to initiate reiterativetemplate copying, and is rather distinguished as a method that relies onsuccessive reuse or novel synthesis of single primer molecules. Themethod that relies on primer substitution may be selected from the groupconsisting of helicase dependant amplification (HDA), exonucleasedependant amplification, recombinase polymerase amplification (RPA), andloop mediated amplification (LAMP). The method that relies on successivereuse or novel synthesis of single primer molecules may be selected fromthe groups consisting of strand displacement amplification (SDA),nucleic acid based amplification (NASBA), and transcription-mediatedamplification (TMA). Depending on the selected amplification method, oneset or two or more sets of primers may be used, wherein the primer maybe a primer for PCR.

The measuring of mRNA expression levels may be a process for diagnosingprognosis of ovarian cancer or a risk of recurrence of ovarian cancer bydetermining the presence of mRNA of GSC, VAV3, FOXA2, LEF1, COMP, AZU1,GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A, or PDK4 and the degree ofexpression thereof in a biological sample of an individual, therebymeasuring amounts of mRNA. As such, mRNA may be measured through directisolation or by using a primer or probe relative to the mRNA. Examplesof analysis methods for the measuring include reverse transcriptionpolymerase chain reaction (RT-PCR), competitive RT-PCR, real-timeRT-PCR, RNase protection assay (RPA), northern blotting, nucleic acidmicroarray including DNA, and any combination thereof. RT-PCR is amethod of analyzing RNA, through PCR, by amplifying cDNA obtained byreverse transcription of mRNA. In RT-PCR, the amplification of cDNA maybe performed using a pair of primers that specifically bind to the genesabove. Following RT-PCR, a band pattern and a band width of a sample maybe identified through electrophoresis to thereby determine theexpression of mRNA of the genes above and the degree of expression.Compared with a control group, the prognosis of ovarian cancer or therisk of recurrence of ovarian cancer in an individual may be easilydetermined. Here, the control group may refer to a normal or negativecontrol group including samples of individuals without ovarian cancer orcompletely cured individuals. The control group may also refer to apositive control group including samples of individuals currentlysuffering from ovarian cancer or experiencing recurrence of ovariancancer.

The term “primer” as used herein may refer to a nucleic acid sequenceincluding a free 3′-terminus hydroxyl group and being capable of forminga base pair with a template complementary to a specific base sequence,and serving as an initiation point for transcription of a templatestrand. The primer may initiate DNA synthesis in the presence of fourdifferent nucleoside triphosphates and a reagent for polymerization in asuitable buffer solution at an appropriate temperature (i.e., DNApolymerase or reverse transcriptase). For example, as a primer specificto mRNA of GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY,FIGF, NKX3-2, WNT3A, or PDK4, a sense and antisense primer set including7 to 50 nucleotide sequences may be used to perform PCR amplification tomeasure an amount of a desired product, thereby identifying theprognosis of ovarian cancer or the risk of recurrence of ovarian cancerof an individual. Conditions for PCR and a length of a sense andantisense primer set may be appropriately selected according totechniques known in the art. The primer may include 10 to 100nucleotides (nts), 15 to 100 nts, 10 to 80 nts, 10 to 50 nts, 10 to 30nts, 10 to 20 nts, 15 to 80 nts, 15 to 50 nts, 15 to 30 nts, 15 to 20nts, 20 to 100 nts, 20 to 80 nts, 20 to 50 nts, or 20 to 30 nts.

The term “probe” as used herein may refer to a nucleic acid fragment,such as an RNA or DNA fragment, specifically binding to a target nucleicacid, such as mRNA, and may be labeled so that the presence of specificmRNA and amounts and expression levels of specific mRNA may bedetermined. The probe may be prepared in the form of an oligonucleotideprobe, a single-stranded DNA probe, a double-stranded DNA probe, or anRNA probe. For example, a probe having a nucleic acid sequencecomplementary to mRNA of GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A,IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A, or PDK4 may be used forhybridization, and then, depending on the degree of hybridization, anamount of mRNA may be measured, thereby identifying the prognosis ofovarian cancer or the risk of recurrence of ovarian cancer of anindividual. Selection of a suitable probe and conditions forhybridization may be appropriately selected according to techniquesknown in the art. The probe may include 10 to 100 nts, 15 to 100 nts, 10to 80 nts, 10 to 50 nts, 10 to 30 nts, 10 to 20 nts, 15 to 80 nts, 15 to50 nts, 15 to 30 nts, 15 to 20 nts, 20 to 100 nts, 20 to 80 nts, 20 to50 nts, or 20 to 30 nts.

In addition, the primer or probe may be chemically synthesized accordingto a solid-phase support synthesis method using phosphoramidite or othersynthesis methods widely known in the art. In addition, the nucleic acidsequence may be modified using various methods known in the art.Examples of such modification include methylation, capping, substitutionof at least one natural nucleotide with an analog thereof, andmodifications between nucleotides, such as modifications with anon-electrically charged linker (e.g., methyl phosphonate,phosphotriester, phosphoramidate, or carbamate) or an electricallycharged linker (e.g., phophorotioate or phosphoroditioate). In addition,the primer or probe may be modified using an indicator capable ofdirectly and/or indirectly providing a detectable signal. Examples ofthe indicator include a radioactive isotope, a fluorescent molecule, andbiotin.

The agent may include a peptide or protein that specifically binds toGSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF,NKX3-2, WNT3A, or PDK4, or mRNA encoding one of these proteins. Thepeptide or protein may include an antibody, a ligand, a receptor, anagonist, an antagonist, or a fragment thereof, or a combination thereof.

The measuring of protein expression levels may be a process fordiagnosing prognosis of ovarian cancer or a risk of recurrence ofovarian cancer by determining the presence of proteins expressed by GSC,VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FGF, NKX3-2,WNT3A, or PDK4, and the degree of expression thereof in a biologicalsample of an individual. As such, an antibody or a fragment thereofcapable of directly isolating GSC, VAV3, FOXA2, LEF1, COMP, AZU1,GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A, or PDK4, or specificallybinding to GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY,FIGF, NKX3-2, WNT3A, or PDK4 may be used to identify GSC, VAV3, FOXA2,LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A, orPDK4. The measurement of the protein analysis may be performed by, forexample, western blotting, enzyme linked immunosorbent assay (ELISA),radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlonyimmunodiffusion, rocket immunoelectrophoresis, tissue immunostaining,immunoprecipitation assay, complement fixation assay,fluorescence-activated cell sorting (FACS), mass spectrometry, magneticbead-antibody immunoprecipitation, a method using a protein chip, or anycombination thereof. For example, various types of ELISA include directELISA in which a labeled antibody immobilized onto a solid support isused to recognize an antigen, indirect ELISA in which a labeled antibodyis used to recognize a capture antibody immobilized on a solid supportand bound to an antigen, direct sandwich ELISA in which a secondaryantibody is used to recognize an antibody which captures an antigencomplexed with a different antibody immobilized onto a solid support, orindirect sandwich ELISA in which a secondary antibody is used torecognize an antibody after the antibody reacts binds to an antigencomplexed with a different antibody immobilized onto a solid support. Inaddition, the expression levels of the proteins may be detected usingsandwich ELISA, which enzymatically develops proteins through attachmentof a labeled antibody that recognizes an antigen complexed with adifferent antibody after immobilizing an antibody on a solid support andallowing a reaction on a sample, or enzymatically develops proteinsthrough attachment of a labeled secondary antibody with respect to anantibody that recognizes an antigen complexed with a different antibody.In this regard, the degree of formation of the protein-antibody complexmay be determined, thereby identifying the prognosis of ovarian canceror the risk of recurrence of ovarian cancer of an individual.

For example, the measurement of the protein expression level may be, forexample, performed by western blotting using at least one antibody withrespect to the protein. Here, all the proteins are separated from asample, are subjected to electrophoresis to separate them by size, andare migrated to a nitrocellulose membrane where they react with anantibody to form an antigen-antibody complex. Afterwards, the amount ofthe proteins is identified in the same manner as in identifying anamount of the antigen-antibody complex by using a labeled antibody, andthus the prognosis of ovarian cancer or the risk of recurrence ofovarian cancer of an individual may be identified.

The method of detecting the protein may include comparing the measuredprotein expression level in the biological sample with that of a normalcontrol group including samples from individuals without ovarian canceror completely cured individuals without any manipulation. Here, the mRNAor protein expression level may be measured according to an absolutevalue of the marker (e.g., μg/ml) or a relative value (e.g., relativeextent of signals).

The term “antibody” as a term known in the art may refer to aspecialized immunoglobulin which is directed toward an antigenic site.The antibody may specifically bind to GSC, VAV3, FOXA2, LEF1, COMP,AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A, PDK4, or a fragmentthereof. Here, a fragment of any of GSC, VAV3, FOXA2, LEF1, COMP, AZU1,GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A, and PDK4 may be, forexample, an immunogenic fragment. Such a fragment may refer to a proteinfragment having at least one epitope corresponding to an antibody forGSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF,NKX3-2, WNT3A, or PDK4. In order to prepare an antibody, GSC, VAV3,FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A,or PDK4 may be cloned into an expression vector, GSC, VAV3, FOXA2, LEF1,COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A, or PDK4encoded by the cloned gene may be obtained, and a common method known inthe art may be performed on the obtained protein.

The antibody may be in the form of a polyclonal antibody, a monoclonalantibody, or a recombinant antibody, and in this regard, all theimmunoglobulin antibodies fall within the range of the antibody of thepresent inventive concept. The antibody may be in a complete formcomposed of two full-length light chains and two full-length heavychains. In addition, the antibody may be a special antibody, such as ahumanized antibody. The polyclonal antibody may be prepared according toa conventional method known in the art by injecting an immunogen (e.g.,a biomarker protein or a fragment thereof) into a foreign host. Theforeign host may include a mammal, such as a mouse, a rat, a sheep, anda rabbit. When the immunogen is injected in an intramuscular,intraperitoneal, or subcutaneous manner, it can be administered with anadjuvant to generally increase antigenicity. Then, blood may beregularly collected from the foreign host to collect serum showingimproved titer and specificity for an antigen, or to separate and purifyantibodies therefrom.

The monoclonal antibody may be prepared by cell line generationtechniques by fusion known to those of skill in the art. For a briefdescription of a method of preparing the monoclonal antibody, Balb/Cmice may be immunized with a suitable amount (e.g., 10 μg) of theprotein that has been purified, or polypeptide fragments of the proteinmay be synthesized and combined with bovine serum albumin to immunizemice, and then, antigen-producing lymphocytes isolated from the mice maybe fused with human or mouse myeloma to produce immortalized hybridomacells. Then, according to ELISA, only hybridoma cells producing desiredmonoclonal antibodies are selected and cultured, and monoclonalantibodies may be separated and purified from the culture. In addition,the monoclonal antibody may be a commercially available antibody forGSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF,NKX3-2, WNT3A, or PDK4.

Such antibodies may be used to identify the protein expression in thebiological sample according to suitable methods known in the art, suchas ELISA, RIA, sandwich assay, or western blotting or immunoblotting ona polyacrylamide gel.

The term “fragment” as used herein may refer to, for example, apolypeptide not having a complete structure of an antibody, peptide, orprotein, but having a binding domain or an antigen-binding site that isspecifically directed toward an antigenic site. The fragment may includea functional fragment of an antibody molecule that is not an antibody ina complete form consisting of two light chains and two heavy chains.Such a fragment of an antibody molecule may refer to a fragment havingat least an antigen-binding function, and may include Fab, F(ab′),F(ab′)₂, or Fv. The binding fragment may include at least 7 amino acids,and for example, at least 9 amino acids or at least 12 amino acids.

The expression level may refer to an expression level in a sampleseparated from an individual, and the sample may be separated fromovaries of the individual. Examples of the sample separated from theindividual include blood, plasma, serum, urine, feces, saliva, tears,cerebrospinal fluid, cells, tissues, and any combination thereof. Theindividual may include a mammal, such as a human, a primate, a dog, acat, a cow, a horse, a pig, a rabbit, or a mouse.

According to one or more embodiments, a kit for diagnosing prognosis ofovarian cancer or a risk of recurrence of ovarian cancer in anindividual includes an agent for measuring an expression level of atleast one gene selected from the group consisting of GSC, VAV3, FOXA2,LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A, andPDK4

The kit may include, for example, a microarray that measures anexpression level of GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F,CD86, PYY, FIGF, NKX3-2, WNT3A, or PDK4, or an expression level of mRNAof a gene encoding these proteins. The microarray may be preparedaccording to a known method in the art using GSC, VAV3, FOXA2, LEF1,COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A, or PDK4. Inthe microarray, mRNA of a gene encoding GSC, VAV3, FOXA2, LEF1, COMP,AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A, or PDK4, or a cDNAhaving a sequence corresponding to a fragment of the gene may beattached as a probe on a substrate.

When the kit is used for, for example, measuring an expression level ofmRNA of GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY,FIGF, NKX3-2, WNT3A, or PDK4, the kit may include necessary elements toperform RT-PCR. Such an RT-PCR kit may include, in addition to primerseach specifically bind to mRNA of marker genes, a test tube or otherproper containers; a reaction buffer solution; deoxynucleotides (dNTPs);enzymes, such as a Taq-polymerase and a reverse transcriptase; DNase; anRNase inhibitor; dEPC-water; or sterilized water. In addition, the kitmay include a pair of primers each specifically binding to a gene usedin a qualitative control group.

In addition, the kit may include a substrate, an appropriate buffer, asecondary antibody labeled with a chromogenic enzyme or a fluorescentsubstance, or a chromogenic substrate, for detecting an antibodyspecifically binding to GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A,IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A, or PDK4, or for immunologicaldetection of antibodies. The substrate may be a nitrocellulose membrane,a 96-well plate synthesized by a polyvinyl resin, a 96-well platesynthesized by a polystyrene resin, or a glass slide. The chromogenicenzyme may be peroxidase or alkaline phosphatase. The fluorescentsubstance may be FITC or RITC. The chromogenic substrate may be2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS),o-phenylenediamine (OPD), or tetramethyl benzidine (TMB).

According to one or more embodiments, a method of diagnosing prognosisof ovarian cancer or a risk of recurrence of ovarian cancer of anindividual for providing necessary information related to diagnosingprognosis of ovarian cancer or a risk of recurrence of ovarian cancer ofan individual includes: forming a complex by contacting a sampleseparated from an individual with one or more materials specificallybinding to one or more proteins selected from GSC, VAV3, FOXA2, LEF1,COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A, and PDK4, orone or more mRNAs encoding any of these proteins; measuring anexpression level of one or more genes selected from GSC, VAV3, FOXA2,LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A, andPDK4 in the sample by measuring a level of the complex; comparing themeasured expression level of the selected one or more genes in thesample with that of the same gene(s) in a control group; and, in thecase of changes in the expression level of the one or more genes in thesample as compared with the control group, determining whether theindividual has poor prognosis of ovarian cancer or a high risk ofrecurrence of ovarian cancer.

The method may include forming a complex by contacting a sampleseparated from an individual with the materials each specificallybinding to GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY,FIGF, NKX3-2, WNT3A, PDK4, or an mRNA encoding any of these proteins.

The expression level may include a protein expression level of GSC,VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2,WNT3A, or PDK4. Thus, the measuring of the expression level may beperformed by measuring an amount of mRNA of GSC, VAV3, FOXA2, LEF1,COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A, or PDK4,proteins, or a combination thereof.

The method may also include measuring an expression level of GSC, VAV3,FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A,or PDK4 in a sample by measuring a level of the complex.

The measuring of the level of the complex may be performed by detectingsignals from a detectable label attached to a material that specificallybinds to any of the proteins above or an mRNA encoding any of theproteins above. The measuring of the level of the complex may be a stepof measuring a level of any of the proteins above or an mRNA encodingany of the proteins above after separating the complex again; a step ofmeasuring a level of the material that specifically binds to any of theproteins above or an mRNA encoding any of the proteins above; or a stepof measuring a level of the complex without separating the complex. Themeasuring of the level of the complex may be performed by RT-PCR,competitive RT-PCR, real-time RT-PCR, RPA, northern blotting, nucleicacid microarray including DNA, western blotting, ELISA, RIA,radioimmunodiffusion, Ouchterlony immunodiffusion, rocketimmunoelectrophoresis, tissue immunostaining, immunoprecipitation assay,complement fixation assay, FACS, mass spectrometry, magneticbead-antibody immunoprecipitation, protein chip, or a combinationthereof.

The method may include comparing the measured expression level of theone or more genes selected from GSC, VAV3, FOXA2, LEF1, COMP, AZU1,GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A, or PDK4 in the samplewith that of the same gene(s) in a control group.

The method may also include determining that the individual has poorprognosis of ovarian cancer or a high risk of recurrence of ovariancancer in the case of higher expression levels of the selected gene(s)in the sample than in the normal or negative control group. In addition,the method may also include determining that the individual does nothave poor prognosis of ovarian cancer or has a low risk of recurrence ofovarian cancer in the case of lower expression levels of the selectedgene(s) in the sample than in the control or negative group.

The method may also include determining, if the expression level of GSC,VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2,WNT3A, or PDK4 in the individual is changed relative to that of the samegene(s) in a control group, whether the individual has poor prognosis ofovarian cancer or has a high risk of recurrence of ovarian cancer. Sucha change in the expression level in the individual may refer to anexpression level similar with that in a normal or negative controlgroup; or an expression level increased by at least 1%, 2%, 3%, 4%, 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%,500%, 600%, 700%, 800%, 900%, or 1,000% as compared with that in anormal or negative control group. In addition, such a change in theexpression level in the individual may refer to an expression levelsimilar with that in a positive control group; or an expression leveldecreased by at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 100% as compared with that in a positive controlgroup.

The method may include evaluating clinicopathologic parameters of apatient, such as chemoresistance, lymph node metastasis, and distantmetastasis of cancer cells, or patient survival.

According to one or more embodiments, a composition for treating ovariancancer or preventing recurrence of ovarian cancer includes an agent toinhibit an expression level of VAV3.

The agent may be effective in inhibition of spheroid-forming capacity ofcancer cells, inhibition of colony formation of cancer cells, inhibitionof proliferation or growth of cancer cells, or enhancement ofsensitivity to other anticancer treatments, thereby directly and/orindirectly treating ovarian cancer or predicting recurrence of ovariancancer. Here, the cancer cells may be CSCs.

The agent may specifically bind to VAV3 or an mRNA sequence encoding theprotein. The agent may inhibit expression of VAV3 by binding to VAV3 orthe mRNA sequence encoding the protein.

The agent may be a nucleotide binding to a sequence of VAV3 in acomplementary manner. The nucleotide may have a length of 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70,80, 90, or 100 nts. The nucleotide may include a sequence havinghomology of at least 100%, 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%,or 70% with the sequence of VAV3, but may also include all sequencesthat can bind to the sequence of VAV3 in a complementary manner. Forexample, the nucleotide may be an siRNA, and the nucleotide may have asequence of SEQ ID NO: 1 or 2.

The agent may be an antibody or a fragment thereof, which specificallybinds to VAV3.

The composition may further include a pharmaceutically acceptablecarrier. The term “pharmaceutically acceptable carrier” as used hereindenotes a material that is used in combination with an activeingredient, that is, generally, an inert material, to help applicationof the active ingredient. Examples of the pharmaceutically acceptablecarrier may include, in general, a pharmaceutically acceptableexcipient, additive, or diluent. Examples of the pharmaceuticallyacceptable carrier may include at least one selected from the groupconsisting of a filler, a binder, a disintegrant, a buffer, apreservative, an antioxidant, a lubricant, a flavoring agent, athickener, a coloring agent, an emulsifier, a suspending agent, astabilizer, and an isotonic agent.

The composition may include the agent for reducing expression level ofVAV3 at a “therapeutically effective amount”. In the composition, theterm “therapeutically effective amount” as used herein refers to asufficient amount that produces a therapeutic effect when administeredto a subject in need of treatment. The term “treatment” as used hereinrefers to treatment of a disease or medical condition, for example,ovarian cancer, including humans, and the meaning of treatment is: (a)preventing the occurrence of the disease or medical condition, that is,by prophylactic treatment of a patient; (b) ameliorating the disease ormedical condition such as by eliminating or causing regression of thedisease or medical condition of a patient; (c) suppressing the diseaseor medical condition such as by slowing or arresting the development ofthe disease or medical condition of a patient; or (d) alleviating asymptom of the disease or medical condition of a patient. Thecomposition may include an “effective amount of an agent for reducingexpression of VAV3 in a cancer cell of a mammal”. The “effective amount”may be appropriately selected by one of ordinary skill in the art. Forexample, the “effective amount” may be in a range of about 0.01 mg toabout 10,000 mg, 0.1 mg to about 1000 mg, about 1 mg to about 100 mg,about 0.01 mg to about 1000 mg, about 0.01 mg to about 100 mg, about0.01 mg to about 10 mg, or about 0.01 mg to about 1 mg. The compositionmay include an agent for reducing expression of VAV3 as an activeingredient. The term “active ingredient” refers to an ingredientenabling a function of the composition as described above, but excludesthe case where the amount of the active ingredient is so small to actlike impurities.

The composition may be prepared for oral administration or parenteraladministration including intravenous, intraperitoneal, subcutaneous,rectal, and topical administration. Thus, the composition may beformulated into various forms such as tablets, capsules, aqueoussolutions, or suspensions. In the case of tablets for oraladministration, an excipient such as lactose or corn starch and alubricant such as magnesium stearate may be added thereto in general. Inthe case of capsules for oral administration, lactose and/or dried cornstarch may be used as a diluent. When an aqueous suspending agent fororal administration is needed, active ingredients may be attached to anemulsifier and/or a suspending agent. If necessary, a predeterminedsweetening agent and/or a flavoring agent may be added to thecomposition. For intraneural, intramuscular, intraperitoneal,subcutaneous, and intravenous administration, a sterilized solution ofthe active ingredients is generally prepared, wherein the pH of thesterilized solution needs to be appropriately adjusted and buffered. Forintravenous administration, the total concentration of solutes needs tobe controlled to render the formulated composition isotonic. Thecomposition may be formulated into an aqueous solution including apharmaceutically acceptable carrier such as salt water at a pH of 7.4.The aqueous solution may be administered to intramuscular or intraneuralblood flow of a patient by local bolus injection.

According to one or more embodiments, a method of treating ovariancancer or preventing recurrence of ovarian cancer includes administeringa composition to an individual, the composition including an agent tolower an expression level of the VAV3 gene.

The method of treating ovarian cancer or preventing recurrence ofovarian cancer may include:

forming a complex by contacting a sample separated from an individualwith a material that specifically binds to at least one protein selectedfrom GSC, VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF,NKX3-2, WNT3A, and PDK4, and an mRNA encoding any of these proteins;measuring expression levels of at least one gene selected from GSC,VAV3, FOXA2, LEF1, COMP, AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2,WNT3A and PDK4 in the sample by measuring levels of the complex;comparing the measured expression levels of the selected gene(s) in thesample with those of the same gene(s) in a control group; anddetermining that the individual has poor prognosis of ovarian cancer ora high risk of recurrence of ovarian cancer in the case of changes inthe expression level of the one or more genes in the sample as comparedwith the control group. In this regard, depending on detection levels ofdisease markers in the individual, pharmaceutically active substancesmay be administered to the individual, thereby providing personalizeddiagnosis and treatment.

According to one or more embodiments, a method of treating ovariancancer or preventing recurrence of ovarian cancer includes:administering a candidate material to an individual; separating a samplefrom the individual, the sample including a target cell; forming acomposition by contacting the separated sample with a material thatspecifically binds to VAV3 or an mRNA encoding the protein; measuring anexpression level of VAV3 in the sample by measuring a level of thecomposition; comparing the measured expression level of the gene in thesample with those of the same gene in a control group; and determining,if the expression level of the gene in the sample is changed relative tothat of the same gene in a control group, the candidate material to havean influence in treating ovarian cancer or preventing recurrence ofovarian cancer.

The method may include administering of the candidate material to theindividual. The candidate material may be a material expected to have aninfluence in treating ovarian cancer or preventing recurrence of ovariancancer. A method of administering the candidate material may beappropriately selected according to a material to be used. For example,the route of administration may be any means, such as intravenous,intramuscular, oral, transdermal, muscosal, intranasal, intratracheal,or subcutaneous administration. The material to be used in the methodmay be administered systemically or locally, and for example, may beadministered to the ovary.

The method may also include determining of, if the expression level ofVAV3 in the individual is changed relative to that of the same gene in acontrol group, the candidate material to have an influence in treatingovarian cancer or preventing recurrence of ovarian cancer. The changesin the expression levels in the individual may refer to expressionlevels increased by at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%,900%, and 1000% as compared with those in a control group; or expressionlevels decreased by at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100% as compared with those in a control group.

Hereinafter, the present inventive concept will be described in detailby explaining preferred embodiments of the inventive concept. However,the preferred embodiments should be considered in descriptive sense onlyand not for purposes of limitation.

Example 1: Selection of Markers of Poor Prognosis of Ovarian Cancer

1. Materials and Experiment Methods

(1) Patients and Tissue Samples

For isolation and evaluation of CSCs, tumor cells of high-grade OSCswere obtained, primarily at the time of surgery, from 17 patients whohad undergone oophorectomy for ovarian carcinoma, and then cultured. Aspheroid formation assay was performed using the cultured primary cancercells.

36 fresh tissue samples of high-grade OSCs were obtained from thepatients, and then, used for qRT-PCR analysis to validate mRNAexpression for differentially expressed genes in the cDNA microarray.The clinicopathological characteristics of the patients are shown inTable 1.

Here, fallopian tubes of patients undergoing hysterectomy withsalpingectomy due to benign myoma uteri were used as healthy controls.

For immunohistochemical (IHC) analysis, formalin-fixed paraffin-embeddedtissues obtained from 74 high-grade OSC patients being treated at theBundang CHA Medical Center were used. The clinicopathologicalcharacteristics of the patients are shown in Table 1.

Here, histologic diagnosis and clinical stages adhere to the WorldHealth Organization (WHO) classification. Histologic stages followed atwo-tiered grading system, and tumor stages followed thetumor-node-metastasis staging (TNM) system.

The samples were divided into a chemosensitive group and achemoresistant group according to responsiveness to first-linechemotherapy (taxol/platinum-based combination therapy) after surgery.

The present study was approved by the Ethics Committee of the BundangCHA Medical Center, and was carried out with informed consent obtainedfrom all patients.

TABLE 1 Clinicopathological characteristics of patients subjected toquantitative real-time PCR analysis and immunohistochemical stainingTotal Chemosensitive Chemoresistant qRT-PCR analysis Age (years) 57.4 ±11.3 56.6 ± 11.3 58.8 ± 11.7 Stage I/II (%)  5 (13.9)  5 (21.7) 0 (0) III/IV (%) 31 (86.1) 18 (78.3) 13 (100)  Nodal metastasis Absent (%) 17(47.2) 11 (47.8)  6 (46.2) Present (%) 19 (52.8) 12 (52.2)  7 (53.8)Distant metastasis Absent (%) 26 (72.2) 16 (69.6) 10 (76.9) Present (%)10 (27.8)  7 (30.4)  3 (23.1) Total (%) 36 23 (68.9) 13 (36.1) IHCanalysis Age (years) 53.8 ± 10.4 52.0 ± 9.8  59.2 ± 10.4 Stage I/II (%)14 (18.9) 14 (25.5) 0 (0)  III/IV (%) 60 (81.1) 41 (74.5) 19 (100) Nodal metastasis Absent (%) 35 (47.3) 28 (50.9)  7 (36.8) Present (%) 39(52.7) 27 (49.1) 12 (63.2) Distant metastasis Absent (%) 38 (51.4) 30(54.5)  8 (42.1) Present (%) 36 (48.6) 25 (45.5) 11 (57.9) RecurrenceAbsent (%) 34 (45.9) 29 (52.7)  5 (26.3) Present (%) 40 (54.1) 26 (47.3)14 (73.7) Total (%) 74 55 (74.3) 19 (25.7) IHC: Immunohistochemistry;qRT-PCR: quantitative real-time polymerase chain reaction

(2) Primary Cell Culture and Spheroid-Forming Cell Isolation

Primary tumor cells were obtained at the time of surgery from 17high-grade OSC patients who had undergone oophorectomy. A tumor mass wascut into small pieces and enzymatically digested into single-cellsuspensions and incubated in Ca²⁺/Mg²⁺-free phosphate-buffered salinecontaining 50 U/mL of collagenase A (Roche, Pleasanton, Calif.). Cellswere incubated with Ber-EP4-coated magnetic Dynabeads (LifeTechnologies, Grand Island, N.Y.) to select epithelial cells, and then,cultured in RPMI medium (Gibcoo/Life Technologies, Grand Island, N.Y.)containing 10% fetal bovine serum, 1% penicillin-streptomycin, and 20ng/mL epidermal growth factor (Life Technologies).

For the spheroid formation assay, single cells were plated on anultralow-attachment 6-well culture plate (Corning, Acton, Mass.) at adensity of 1×10³ cells/cm² in a serum-free Dulbecco's modified Eagles'smedium (Life Technologies) supplemented with 20 ng/mL epidermal growthfactor (Life Technologies), 10 ng/mL basic fibroblast growth factor(Sigma-Aldrich, St Louis, Mo.), 0.4% bovine serum albumin(Sigma-Aldrich), and 5 μg/mL insulin (Sigma-Aldrich). Spheroid formationof 50-100 cells was assessed at 7 days after seeding. Thespheroid-forming efficiency was defined as the ratio of colonies/cellsseeded per well.

(3) cDNA Microarray Analysis

The cDNA microarray was performed on four spheroid-forming cell (SFC)samples and corresponding primary cancer cells.

CSCs were obtained using the iScript cDNA synthesis kit (Bio-Rad,Reymond, Wash.), and synthesis of target cDNA probes and hybridizationwere performed using the Agilent's Low RNA Input Linear Amplificationkit (Agilent Technology, Santa Clara, Calif.). Amplified and labeledcRNAs were purified on the cRNA Cleanup Module (Agilent Technology). Thefragmented cRNAs were directly pipetted onto assembled Human OligoMicroarrays (60K) (Aligent Technology).

The hybridized images were scanned using a DNA microarray scanner, andthen, quantified with Feature Extraction Software (Agilent Technology).Data normalization and selection of significantly changed genes wereperformed using GeneSpring GX 7.3 (Agilent Technology). The averages ofnormalized ratios were calculated by dividing the average normalizedsignal channel intensity by the average normalized control channelintensity. Functional annotation of genes was performed according to theGene Ontology Consortium (www.geneontology.org/index.shtml) byGeneSpring GX 7.3. Gene classification was performed based on DAVID(http://david.abcc.ncifcrf.gov/) and Medline database(www.ncbi.nlm.nih.gov/).

(4) Qualitative Real-Time PCR

Total RNA was isolated from tissues or primary cells using TRIzolreagent (Life Technologies). Synthesis of first-strand cDNA wasperformed using the Superscript III kit (Life Technologies). Real-timePCR was performed in triplicate using the CFX96 real-time PCR detectionsystem (Bio-Rad Laboratories, Hercules, Calif.). 20 μL of the finalreaction product included 0.5 μL of cDNA template, 10 μL of TaqMAnMaster Mix (Applied Biosystems, Paisley, United Kingdom), and 1 μL of amixture of primers and probes. Transcript levels were normalized versusglyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. The geneexpression was calculated using the formula 2^(−ΔΔCt).

(5) Tissue Microarray and IHC Staining

For the IHC analysis, 74 OSC samples were used to construct a tissuemicroarray. For each sample, two tissue cores with diameters of 3 mmwere punched out from each tissue mass, and then, arranged into paraffinblocks using a manual microarray device (UNITMA; Quick-RAY™ UNITechScience, Seoul, Korea). Tissue microarray paraffin sections weredeparaffinized in xylene. Endogenous peroxidase activity was blockedwith 0.3% hydrogen peroxide. For antigen retrieval, the sections wereheated in 0.1 M citrate buffer (pH 6.0) for 15 minutes in a microwaveoven.

Slides were incubated overnight at a temperature of 4° C. with thefollowing primary antibodies and working dilutions: FOXA2 (1:200; Abcam,Cambridge, United Kingdom), LEF1 (1:250; Abcam), PYY (1:50; Abcam), VAV3(1:50; Novus Biologicals, Littleton, Colo.), NKX3-2 (1:75; NovusBiologicals), and Wnt3A (1:750; Novus Biologicals). The slides were thenincubated with a secondary antibody for 15-30 minutes using the HRPPolymer Ultravision LP Detection System (Thermo Scientific, Waltham,Mass.) at room temperature. The sections were then developed withdiaminobenzidine and counterstained with hematoxylin. The IHC stainswere interpreted by two independent pathologists.

Protein expression was determined by an immunoreactive score. Thepercentage of positive cells was scored as 0 (negative), 1 (<10%positive cells), 2 (<10-50% positive cells), 3 (<51-75% positive cells),or 4 (>75% positive cells). The staining intensity was categorized as 0(negative), 1 (weak), 2 (moderate), or 3 (strong). The finalimmunoreactive score was calculated by multiplying these two scores.

(6) Functional Assay for VAV3

Transfection of VAV3 siRNA

For the functional assay for VAV3, paclitaxel (PTX)-resistant SKpaccells, which were generated by continuous exposure of SKOV3 (ATCC,Manassas, Va.) cells to a stepwise escalating concentration of PTX over8 months, were used. VAV3 siRNA and negative siRNA (as a normal controlgroup) were synthesized by Invitrogen (Carlsbad, Calif.). The day beforetransfection, 1×10⁵ cells were seeded into each well of a six-wellplate. The next day, the cells were transfected with annealed siRNAoligos using Lipofectamine 2000 (Invitrogen) according to themanufacturer's instructions. At 24 hours and 48 hours aftertransfection, the cells were harvested, and the efficiency of VAV3knockdown after siRNA transfection was confirmed by qRT-PCR. The cellswere then prepared for MTT, colony formation, and spheroid formationassays.

(7) MTT Analysis for Cell Viability

Cells (1×10⁴) were seeded into a 96-well culture plate, and subsequentlytreated with 10 pmol VAV3 siRNA in a culture medium for 24 hours and 48hours. Control cells were treated with 10 pmol negative siRNA in aculture medium. After 24 hours and 48 hours, the cells were incubatedwith MTT reagent (0.5 mg/mL) at a temperature of 37° C. for 4 hours. Theresulting formazan crystals were solubilized by the addition of 100 μLDMSO to each well.

(8) Colony Formation Assay

Cells were seeded into each well of a 6-well plate at a density of 1×10⁵cells per well. The next day, the cells were transfected with siRNA, andthen, incubated for 48 hours. Transfected cells were then replated in agelatin-coated 6-well culture dish at a density of 300 cells per well.After 14 days, colonies were visualized using hematoxylin after fixationwith 4% paraformaldehyde for 10 minutes. Groups including more than 50cells were scored as colonies.

(9) Statistical Analysis

Statistical analysis was conducted using the SPSS statistics softwarepackage (IBM SPSS Statistics Data Editor 20). The relations between mRNAand protein expression levels in each tumor group and clinicopathologicfactors were evaluated by χ² analysis, Fisher's exact test, or theMann-Whitney test. For survival analysis, the Kaplan-Meier method andthe Cox proportional hazards model were used. The results of eachfunctional assay are expressed as mean±standard error. The Student'st-test was performed and P<0.05 was considered significant.

2. Screening of SFC-Specific Biomarkers to Assess Pathologic Status ofOvarian Cancer

(1) Confirmation of Presence of Large Amounts of CSCs within SFCsIsolated from Cultured Primary Cells from Human Ovarian Carcinomas(OCSs)

The non-adherent spherical clusters of 50-100 cells were observed 1 weekafter plating for the spheroid formation assay (see FIG. 1A). The SFCswere collected, and then, the spheroid-forming capacity thereof wasassessed. The efficiency of spheroid formation from the inoculated cellswas about 2.37%±0.4% in the first generation. These floating sphereswere enzymatically dissociated, and single cells were harvested and usedto form secondary spheroids under the same culture conditions. Thespheroid-forming capacity in the second generation was similar to thefirst generation (2.17%±0.3%) (see FIG. 1B and Table 2).

TABLE 2 Primary and secondary spheroid-forming capability of OCSsPrimary (%) Secondary (%) SCN1 2.82 2.21 SCN2 2.42 2.2 SCN3 2.55 2.05SCN4 3.01 2.12 SCN5 2.45 2.31 SCN6 2.33 2.3 SCN7 2.01 1.98 SCN8 2.3 2.19SCN9 1.98 1.97 SCN10 1.88 1.8 SCN11 2.17 2.1 SCN12 1.89 1.73 SCN13 3.253.06 SCN14 1.79 1.75 SCN15 2.57 2.34 SCN16 2.19 2.14 SCN17 2.7 2.61 Mean± SD 2.37 ± 0.4 2.17 ± 0.3 SCN: serous carcinoma; SD: standard deviation

To examine whether the CSCs were enriched in SFCs, the mRNA expressionof the well-known stem cell marker was analyzed in the SFCs by qRT-PCR,and the analysis results are compared with those of the parental primarycancer cells. The stem cell markers being assessed herein are ALDH1,CD24, CD44, CD133, NOTCH3, SOX2, and CD117. All of these stem cellmarkers, except CD117, were more highly expressed in the SFCs than inthe primary cancer cells (P<0.05). Thus, it was confirmed that the CSCswere enriched in the SFCs (see FIG. 1C), and such SFCs were referred asCSC-like cells.

(2) Confirmation of Gene Expression Profiles of CSC-Like Cells by cDNAMicroarray

The cDNA microarray was performed to assess the gene expression profileof the spheroid-forming CSC-like cells. Gene expression was comparedbetween four samples of CSC-like cells and their corresponding parentalprimary cancer cells. Consequently, it was confirmed that the expressionof 619 genes was significantly increased or decreased at least 5-fold inthe CSC-like cells as compared with the control cancer cells (P<0.05).Among these genes, 381 exhibited increased expression, and 238 exhibiteddecreased expression. In particular, hierarchical clustering of 62 genesthat were significantly altered more than 15-fold in the CSC-like cellsis shown in FIG. 2A. The microarray data presented herein were preparedaccording to the Minimum Information About a Microarray Experiment(MIAME) recommendations, and are accessible through the Gene ExpressionOmnibus (GEO) Series accession number GSE60765(www.ncbi.nlm.nih.gov/geo).

The genes that were increased or decreased in their expression by atleast 2-fold in the CSC-like cells were classified according tobiological process gene ontology. FIG. 2B provides a pie chart showingthe proportion of genes representing each process. Referring to FIG. 2B,the genes present in the greatest number were associated with apoptosisand proliferation (13% each); followed by cell cycle (10%); CSC (8%) andsignal transduction (8%); transforming growth factor-β (TGF-β) (6%);mitogen-activated protein kinase (MAPK) (6%); tyrosine kinase (6%) andNotch signaling (6%); drug response (5%); and Wnt signal transduction(5%); hedgehog signaling (4%); epithelial-mesenchymal transition (3%)and Toll-like signaling (3%); and insulin receptor pathway (2%).

Among the CSC markers, CD24 (13.85-fold), ALDH1A1 (6.80-fold), and SOX2(5.36-fold) were significantly upregulated in spheroid-forming CSC-likecells. The oncogenesis-related genes, which were increased more than5-fold in CSC-like cells as compared with the control cancer cells, andtheir associated functions are summarized in Table 3.

TABLE 3 Genes known to be related with oncogenesis and upregulated morethan 5-fold in CSCs as compared with primary cancer cells Relative GenesFunction fold P-value AGT Angiotensinogen (serpin peptidase inhibitor,clade A, member 8) SIG 7.13 <0.05 ALDHIA1 Aldehyde dehydrogenase 1family, member A1 CSC 6.8 <0.05 ANK2 Ankyrin 3, mode of Ranvier (ankyrinG) SIG 8.27 <0.05 AZU1 Azurocidin 1 APO 13.36 <0.05 CD24 CD24 moleculeCSC 13.85 <0.05 CD86 CD86 molecule PRO 8.67 <0.05 COL15A1 Collagen, typeXV, alpha 1 SIG 8.95 <0.05 COMP Cartilage oligomeric matrix protein APO15.34 <0.05 DNAH17 Dynein, axonemal, heavy chain 17 SIG 5.64 <0.05 EGR3Early growth response 3 SCT 5.36 <0.05 FIGF c-fos-induced growth factor(vascular endothelial growth PRO, SIG, NOT 14.01 <0.05 factor D) FOXA2Forkhead box A2 SIG, SCT 15.11 <0.05 GRIN2A Glutamate receptor,ionotropic, N-methyl-o-aspartate 2A SIG, DRU 7.34 <0.05 GSC Goosecoidhomeobox EMT 8.39 <0.05 HEYL Hairy/enhancer of split related with YRPWmotif-like EMT, NOT 5.36 <0.05 HGF Hepatocyte growth factor (hepapoietinA; scatter factor) EMT, APO, PRO, 6.66 <0.05 CYC, SIG IL17F Interleukin17F SIG 17.36 <0.05 LEF1 Lymphoid enhancer-binding factor 1 EMT, APO,28.76 <0.05 SIG, WNT MMP10 Matrix metallopeptidase 10 (stromelysin 2)SIG 25.87 <0.05 NKX3-2 NK3 homeobox 2 APO 9.43 <0.05 NR6A1 Nuclearreceptor subfamily 6, group A, member 1 PRO 5.15 <0.05 NTRK2Neurotrophic tyrosine kinase, receptor, type 2 TYR 5.76 <0.05 NUMB Numbhomolog (Drosophila) NOT 9.13 <0.05 OR10A7 Olfactory receptor, family10, subfamily A, member 7 SIG 20.94 <0.05 ORIN2 Olfactory receptor,family 1, subfamily N, member 2 SIG 5.15 <0.05 OR2T4 Olfactory receptor,family 2, subfamily T, member 4 SIG 7.02 <0.05 OR9G4 Olfactory receptor,family 9, subfamily G, member 4 SIG 5.37 <0.05 PDGFRB Platelet-derivedgrowth factor receptor, beta polypeptide TYR 5.4 <0.05 PDK4 Pyruvatedehydrogenase kinase, isozyme 4 INS, TYR 11.83 <0.05 POU4F1 POU class 4homeobox 1 SCT 6.84 <0.05 PROC Protein C (inactivator of coagulationfactors, Va and VIIIa) APO 6.74 <0.05 PYY Peptide YY PRO 11.98 <0.05RASD1 RAS, dexamethasone-induced 1 SIG 5.79 <0.05 SERPINF1 Serpinpeptidase inhibitor, clade F, member 1 PRO 6.41 <0.05 SOX2 SRY(sex-determining region Y)-box 2 CSC, SCT 5.36 <0.05 SPEN Spen homolog,transcriptional regulator (Drosophila) NOT, TYR, WNT 5.64 <0.05 SYCE1Synaptonemal complex central element protein 1 CYC 8 <0.05 TNFSF10 Tumornecrosis factor (ligand) superfamily, member 10 SIG, NOT, TGF 6.72 <0.05TRIL TLR4 interactor with leucine-rich repeats TOL 7.19 <0.05 UCN2Urocortin 2 PRO 6.95 <0.05 VAV3 vav 3 guanine nucleotide exchange factorSIG 11.75 <0.05 WNT3A Wingless-type MMTV integration site family, member3A PRO, WNT, HED 6.19 <0.05 APO: apoptosis; CSC: cancer stem cellmarker; CYC: cell cycle; DRU: drug response; EMT: epithelial mesenchymaltransformation; HED: hedgehog pathway; INS: insulin receptor; NOT: Notchsignaling pathway; PRO: proliferation; SCT: stem cell transcription;SIG: signal transduction; TGF: transforming growth factor-β pathway;TOL: Toll-like receptor signaling pathway; TYR: tyrosine kinasesignaling; WNT: Wnt pathway.

(3) Selection of 14 Genes as Candidates for Biomarkers and Validation byqRT-PCR

To identify candidate biomarkers for predicting poor prognosis andchemoresistance, 14 genes were selected based on the results of the cDNAmicroarray and gene ontology. In particular, genes upregulated more than5-fold in CSC-like cells by cDNA microarray were identified, and then,the most highly expressed genes associated with oncogenesis wereselected therefrom. The relative expression levels of these 14 geneswere as follows: GSC (8.39-fold), VAV3 (11.75-fold), FOXA2 (15.11-fold),LEF1 (28.76-fold), COMP (15.34-fold), AZU1 (13.36-fold), GRIN2A(7.34-fold), IL17F (17.36-fold), CD86 (8.67-fold), PYY (11.98-fold),FIGF (14.01-fold), NKX3-2 (9.43-fold), WNT3A (6.19-fold), and PDK4(11.83-fold).

In addition, the mRNA expression levels of these 14 genes in the SFCsand their parental cancer cells were validated by qRT-PCR. Consequently,it was confirmed that GSC (4.26-fold; P<0.001), VAV3 (7.05-fold;P<0.001), FOXA2 (12.06-fold; P<0.05), LEF1 (17.26-fold; P<0.05), COMP(21.33-fold; P<0.001), GRIN2A (9.36-fold; P<0.001), CD86 (23.14-fold;P<0.001), PYY (4.18-fold; P<0.001), NKX3-2 (10.35-fold; P<0.001), andPDK4 (74.26-fold; P<0.001) were significantly upregulated in SFCs ascompared with parental cancer cells (see FIG. 2C).

(4) Validation of Increased Expression Levels of LEF1, PYY, NKX3-2, andWNT3A in Chemoresistant Cancer Cells

To assess the mRNA expression of the candidate genes, qRT-PCR wasperformed on 36 fresh human OSC tissues. Then, the mRNA expressionlevels in the OSC tissues were compared with those in normal fallopiantubes (controls).

Consequently, it was confirmed that the following 11 genes weresignificantly overexpressed in the human OSCs: VAV3 (7.92-fold), FOXA2(7.32-fold), LEF1 (9.64-fold), COMP (25.01-fold), GRIN2A (12.51-fold),IL17F (2.50-fold), CD86 (6.35-fold), PYY (13.50-fold), NKX3-2(10.79-fold), WNT3A (15.67-fold), and PDK4 (10.19-fold) (all P<0.05).

FIG. 3A shows the relative expression of 13 candidate genes in the OSCsand controls. Referring to FIG. 3A, LEF1, PYY, NKX3-2, and WNT3A showedsignificant differences in their expression levels between OSCs andcontrols (P<0.05). In particular, the expression level of LEF1 wasincreased about 2.85-fold, the expression level of PYY was increasedabout 20.03-fold, the expression level of NKX3-2 was increased about3.02-fold, and the expression level of WNT3A was increased about3.91-fold in the OSCs, which are chemoresistant cancer cells, ascompared with chemosensitive cells (controls) (see FIG. 3B).

Furthermore, the correlation between the mRNA expression levels andclinicopathologic parameters was analyzed, and it was confirmed that theincreased expression of VAV3 (>2-fold relative to normal controls) wasassociated with distant metastasis (P<0.05). Likewise, the increasedexpression of PYY (>2-fold relative to normal controls) was associatedwith lymph node metastasis (P<0.05) and chemoresistance (P<0.05), andthe increased expression of FoxA2 (>6-fold relative to normal controls)and NKX3-2 (>10-fold relative to normal controls) were associated withchemoresistance (P<0.05) (see FIG. 3C).

(5) Validation of Correlation Between Overexpression of VAV3, NKX3-2,and LEF1 in OSCs and Poor Prognosis

An IHC study was performed on a tissue microarray including 74 OSCtissue samples to validate the protein expression of the candidate genesand to assess the association between the protein expression of thesegenes and clinicopathologic parameters, such as clinical state, lymphnode, distant metastasis, chemoresistance, and survival. As targets forthe study, six proteins, i.e., LEF1, PYY, NKX3-2, WNT3A6, VAV3, andFOXA2, were selected. When the immunoreactive score was >4 for LEF1,NKX3-2, PYY, and Wnt3A; >2 for FOXA2; and 26 for VAV3, the samples wereconsidered as exhibiting overexpression.

Consequently, staining was observed in the cytoplasm or the nucleus ofthe OSC tissue, focally or diffusely (see FIG. 4A). In particular,FOXA2, LEF1, and NKX3-2 showed nuclear positivity, PYY showedcytoplasmic positivity, and VAV3 showed both nuclear and cytoplasmicpositivity.

The epithelial and stromal cells of benign serous cystadenomas andnormal fallopian tubes were negative or weakly positive (less than 5%positive cells) for FOXA2, LEF1, NKX3-2, and VAV3.

Apical staining for Wnt3A was found in epithelial cells of normalfallopian tubes and benign serous cystadenomas, whereas membranousstaining was observed in OSC tissues.

The majority of OSCs overexpressed these proteins. In particular, FOXA2was overexpressed in 66.2% (49/74) of cases; LEF1 was overexpressed in59.5% (44/74) of cases; and PYY, NKX3-2, and Wnt3A were overexpressed in58.1% (43/74), 58.1% (43/74), and 63.5% (47/74) of cases, respectively.The relationships between IHC staining and clinicopathologic parametersare shown in Table 4. Referring to Table 4, distant metastasis wassignificantly related to the expression of LEF1, VAV3, and NKX3-2(P<0.05), and chemoresistance was significantly related to theoverexpression of NKX3-2 (P<0.05).

TABLE 4 Relationships between immunohistochemical expression andclinicopathologic parameters in OSCs (N = 74) FOXA2 LEF1 VAV3 PYY NKX3-2Wnt3A OE P-value OE P-value OE P-value OE P-value OE P-value OE P-valueAge  <55 24/42 0.059 24/42 0.642 31/42 0.668 25/42 0.777 22/42 0.25328/42 0.519 ≧55 25/32 20/32 25/32 18/32 21/32 19/32 Clinical stage I andII  9/14 0.865  7/14 0.423  9/14 0.270  7/14 0.495  5/14 0.059 10/140.494 III and IV 40/60 37/60 47/60 36/60 38/60 37/60 Nodal metastasisAbsent 23/35 0.931 19/35 0.390 10/35 0.420 22/35 0.433 19/35 0.528 20/350.281 Present 26/39 25/39 31/39 21/39 24/39 27/39 Distant metastasisAbsent 24/38 0.568 18/38 <0.05^(a) 23/38 <0.05^(a) 24/38 0.366 17/38<0.05^(a) 23/38 0.583 Present 25/36 26/36 33/36 19/36 26/36 24/36Chemoresistance Sensitive 35/55 0.425 31/55 0.356 42/55 0.814 33/550.575 28/55 <0.05^(a) 35/55 0.970 Resistance 14/19 13/19 14/19 10/1915/19 12/19 ^(a)p < 0.05. A sample was considered OE (overexpression)when the immunoreactive score was ≧2 for FOXA2, ≧4 for LEF1, PYY,NKX3-2, and Wnt3; and ≧6 for VAV3.

Example 2. Validation of Effects of VAV3 Knockdown Using VAV3sIRNA inCSCs

(1) Validation of Associations Between High VAV3 Immunoreactivity andPoor Prognosis in OSCs

A survival analysis was performed for 74 OSC patients according to theirprotein expression. Experiment materials and methods used herein werethe same as those described in connection with Example 1. The proteinexpression was analyzed by immunohistochemistry. The median follow-upperiod was 31 months (1-112 months). During the follow-up period, 21patients (28.4%) died of disease. According to the Kaplan-Meieranalysis, it was found that the overexpression of VAV3 (immunoreactivescore ≧6) and FOXA2 (immunoreactive score ≧2) was significantlyassociated with short overall survival (OS) (VAV3, OS: 64.2% inoverexpressed cells vs. 94.4% in controls, P<0.05; FOXA2, OS: 63.3% inoverexpressed cells vs. 88% in controls, P<0.05). The expression ofother proteins was not associated with patient survival.

A multivariate Cox regression analysis was performed forclinicopathological parameters and the immunoreactivity of VAV3 andFOXA2. The overexpression of VAV3 was found to be an independentindicator of poor prognosis in OSC patients (see Table 5; hazard ratio:15.27, P<0.05). However, the overexpresison of FOXA2 was notsignificantly associated with OS in the present analysis. In addition,chemoresistance was significantly associated with poor survival (hazardratio: 17.74, P<0.001)

TABLE 5 Multivariate Cox regression analysis for clinicopathologicparameters and immunohistochemical expression of VAV3 and FOXA2 No. ofcases Overall Hazard (n = 74) Death survival ratio P-value Age <55 42 935.0 ± 29.7 2.38 0.134 ≧55 32 12 26.5 ± 23.1 Clinical stage I/II 14 138.8 ± 24.2 3.83 0.220 III/IV 60 20 29.6 ± 27.7 Distant metastasisAbsent 38 10 38.6 ± 27.1 1.10 0.877 Present 36 11 23.6 ± 25.4Chemoresistance Sensitive 55 13 37.0 ± 29.1 17.74 <0.05^(a) Resistant 198 15.0 ± 9.1  VAV3 expression Low 18 1 33.1 ± 27.5 15.27 <0.05^(a) High56 20 30.8 ± 27.3 FOXA2 expression Low 25 3 33.0 ± 30.5 2.51 0.222 High49 18 30.5 ± 25.6 ^(a)P < 0.05.

(2) Effect of Inhibition of VAV3 Knockdown on Spheroid Formation andValidation of Decreased Cancer Cell Viability and Proliferation

1) Spheroid Formation Assay

With VAV3 siRNA (SEQ ID NO: 1 or 2) treatment (i.e., addition of VAV3siRNA to cancer cells), VAV3 knockdown cells were compared with controlswith respect to their number and size of spheroids to validate theeffect of VAV3 siRNA on CSC activation by VAV3 siRNA.

Consequently, the number of the spheroids formed in CSCs significantlydecreased after the treatment with VAV3 siRNA (30% decrease comparedwith negative siRNA controls, P<0.001), and the size of the spheroidswas also significantly decreased (see FIG. 5A). That is, it wasconfirmed that VAV3 knockdown inhibited CSC activation.

2) Colony Formation Assay

According to the colony formation assay, it was confirmed that clonalgrowth of SKpac cells was significantly inhibited by VAV3 siRNA. Inparticularly, the number of colonies of SKpac cells treated with VAV3siRNA decreased by 41% as compared with the control cells treated withnegative siRNA (P<0.001) (see FIG. 5B).

3) PTX Drug Sensitivity Assay

The effects of VAV3 inhibition on sensitivity to PTX of PTX-resistantSKpac cells were assessed. In particular, the cell viability wasexamined using an MTT assay.

Consequently, it was confirmed that the number of SKpac cells decreasedby 27% and 16% at 24 hours and 38 hours, respectively, (P<0.05) upon theaddition of VAV3 siRNA+PTX, as compared with the addition ofPTX+negative siRNA (controls) (see FIG. 5C).

As described above, according to the one or more of the above exampleembodiments of the present inventive concept, a composition, a kit, anda method of diagnosing prognosis of ovarian cancer or a risk ofrecurrence of ovarian cancer of an individual may be used efficiently.According to the one or more of the above example embodiments of thepresent inventive concept, a composition may be used efficiently totreat ovarian cancer or prevent recurrence of ovarian cancer. Accordingto the one or more of the above example embodiments of the presentinventive concept, a method of screening a material for treating ovariancancer or preventing recurrence of ovarian cancer may be usedefficiently to select a candidate material capable of treating ovariancancer or preventing recurrence of ovarian cancer.

It should be understood that embodiments described herein should beconsidered in a descriptive sense only and not for purposes oflimitation. Descriptions of features or aspects within each embodimentshould typically be considered as available for other similar featuresor aspects in other embodiments.

While one or more embodiments have been described with reference to thefigures, it will be understood by those of ordinary skill in the artthat various changes in form and details may be made therein withoutdeparting from the spirit and scope of the disclosure as defined by thefollowing claims.

What is claimed is:
 1. A method of predicting prognosis of ovariancancer or a risk of recurrence of ovarian cancer of an individual, themethod comprising: forming a complex by contacting a sample separatedfrom an individual with one or more materials specifically binding toone or more proteins selected from GSC, VAV3, FOXA2, LEF1, COMP, AZU1,GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A, and PDK4, or one or moremRNAs encoding any of these proteins; measuring an expression level ofone or more genes selected from GSC, VAV3, FOXA2, LEF1, COMP, AZU1,GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A, and PDK4 in the sample bymeasuring a level of the complex; comparing the measured expressionlevel of the selected one or more genes in the sample with that of thesame gene(s) in a control group; and, in the case of changes in theexpression level of the one or more genes in the sample as compared withthe control group, determining whether the individual has poor prognosisof ovarian cancer or a high risk of recurrence of ovarian cancer.
 2. Themethod of claim 1, wherein the forming of the complex comprisescontacting the sample with materials each specifically binding to VAV3,FOXA2, LEF1, NKX3-2, or WNT3A, or an mRNA encoding any of theseproteins.
 3. The method of claim 1, wherein the one or more materialsare each a primer, a probe, a nucleotide, an antibody or anantigen-binding fragment thereof, a ligand, a receptor, an agonist or anantagonist, a protein, or any combination thereof, each of which bindsto one or more proteins selected from GSC, VAV3, FOXA2, LEF1, COMP,AZU1, GRIN2A, IL17F, CD86, PYY, FIGF, NKX3-2, WNT3A, and PDK4, or one ormore mRNAs encoding any of these proteins.
 4. The method of claim 1,wherein the method comprises evaluating chemoresistance of cancer cells,lymph node metastasis, distant metastasis, or survival of a patient. 5.The method of claim 1, wherein the measuring of the expression level isperformed by a reverse transcription polymerase chain reaction (RT-PCR),competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA),northern blotting, nucleic acid microarray using DNA, western blotting,enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA),radioimmunodiffusion, Ouchterlony immunodiffusion, rocketimmunoelectrophoresis, tissue immunostaining, immunoprecipitation assay,complement fixation assay, fluorescence-activated cell sorting (FACS),mass spectrometry, magnetic bead-antibody immunoprecipitation, proteinchip, or any combination thereof.
 6. A method of treating ovarian canceror preventing recurrence of ovarian cancer, the method comprising:administering a composition to an individual, the composition comprisingan agent that inhibits gene expression level of VAV3.
 7. The method ofclaim 6, wherein the agent is a VAV3 siRNA that targets an mRNA encodingVAV3.
 8. The method of claim 7, wherein the VAV3 siRNA has a nucleicacid sequence of SEQ ID NO: 1 or
 2. 9. The method of claim 6, whereinthe composition enhances sensitivity of cancer cells to other anticancertreatments.
 10. The method of claim 6, wherein the composition inhibitsformation of spheroids of cancer cells.
 11. A method of screening amaterial treating ovarian cancer or preventing recurrence of ovariancancer, the method comprising: administering a candidate material to anindividual; separating a sample from the individual, the samplecomprising a target cell; forming a complex by contacting the separatedsample with a material specifically binding to VAV3 or an mRNA encodingVAV3; measuring an expression level of VAV3 in the sample by measuring alevel of the complex; comparing the measured expression level of thegene in the sample with that of the same gene in a control group; and,in the case of changes in the expression level of the gene in the sampleas compared with the control group, determining whether the candidatematerial is efficient in treating ovarian cancer or preventingrecurrence of ovarian cancer.
 12. The method of claim 11, wherein thematerial specifically binding to VAV3 or an mRNA encoding VAV3 is aprimer, a probe, a nucleotide, an antibody or an antigen-bindingfragment thereof, a ligand, a receptor, an agonist or an antagonist, aprotein, or any combination thereof.